The present invention relates to recombinant DNA encoding the AsiSI restriction endonuclease (endonuclease) as well as AsiSI methyltransferase (methylase), expression of AsiSI restriction endonuclease and methylase in E. coli cells containing the recombinant DNA.
AsiSI endonuclease is found in the strain of Arthrobacter species S (New England Biolabs"" strain collection #1221). It recognizes the double-stranded DNA sequence 5xe2x80x2GCGAT/CGC3xe2x80x2 (SEQ ID NO:1) and cleaves between the T and C to generate a 2-base 3xe2x80x2 overhanging ends (/indicates the cleavage of phosphodiester bond). AsiSI methylase (M.AsiSI) is also found in the same strain. It recognizes the double-stranded DNA sequence 5xe2x80x2GCGATCGC 3xe2x80x2 (SEQ ID NO:1) and presumably modifies the C5 position of cytosine at base number 2, or number 6, or number 8. The 5mC modified AsiSI site is resistant to AsiSI restriction digestion.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial/viral proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases recognize and bind particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) on DNA molecules. Once bound, they cleave the molecule within (e.g. BamHI), to one side of (e.g. SapI), or to both sides (e.g. TspRI) of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are discovered. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2 (SEQ ID NO:2), 5xe2x80x2PuG/GNCCPy3xe2x80x2 (SEQ ID NO:29) and 5xe2x80x2CACNNN/GTG3xe2x80x2 (SEQ ID NO:3) respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2 (SEQ ID NO:4).
A second component of bacterial/viral restriction-modification (R-M) systems are the methylase. These enzymes co-exist with restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. Only unmodified, and therefore identifiably foreign DNA, is sensitive to restriction endonuclease recognition and cleavage. During and after DNA replication, usually the hemi-methylated DNA (DNA methylated on one strand) is also resistant to the cognate restriction digestion.
With the advancement of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop an efficient method to identify such clones within genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted clones with non-methylase inserts are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloning method used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78: 1503-1507, (1981)). Since the expression of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phage. However, this method has been found to have only a limited success rate. Specifically, it has been found that cloned restriction-modification genes do not always confer sufficient phage resistance to achieve selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning vectors (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421, (1985)). Since restriction-modification genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baidauf, Gene 21:111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of thermostable restriction endonuclease genes into E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response signals following DNA damage caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535). The disadvantage of this method is that sometimes positive blue clones containing a restriction endonuclease gene are difficult to culture due to the lack of the cognate methylase gene.
There are three major groups of DNA methylases based on the position and the base that is modified (C5 cytosine methylases, N4 cytosine methylases, and N6 adenine methylases). N4 cytosine and N6 adenine methylases are amino-methyltransferases (Malone et al. J. Mol. Biol. 253:618-632, (1995)). When a restriction site on DNA is modified (methylated) by the methylase, it is resistant to digestion by the cognate restriction endonuclease. Sometimes methylation by a non-cognate methylase can also confer the DNA site resistant to restriction digestion. For example, Dcm methylase modification of 5xe2x80x2CCWGG3xe2x80x2 (W=A or T) (SEQ ID NO:5) can also make the DNA resistant to PspGI restriction digestion. Another example is that CpG methylase can modify the CG dinucloetide and make the NotI site (5xe2x80x2GCGGCCGC3xe2x80x2 (SEQ ID NO:6)) refractory to NotI digestion (New England Biolabs"" Catalog, 2000-01, page 220). Therefore methylases can be used as a tool to modify certain DNA sequences and make them uncleavable by restriction enzymes.
Because purified restriction endonucleases and modification methylases are useful tools for creating recombinant molecules in the laboratory, there is a great commercial interest to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such over-expression strains should also simplify the task of enzyme purification.
The present invention relates to a method for cloning the AsiSI restriction endonuclease from Arthrobacter species S into E. coli by methylase selection and inverse PCR amplification of the adjacent DNA.
The cloning of AsiSI endonuclease and methylase genes proved to be extremely difficult. No M.AsiSI positive clones were identified in a Sau3AI partial genomic DNA library. Neither were M.AsiSI positive clones identified in AatII, AccI, Af/III, KasI, NdeI, and XmaI genomic libraries using methylase selection. The difficulty may result from poor expression of asiSIM gene in E. coli and/or cross contamination of other common cloning vectors such as pUC18, pUC19, and pBR322 (ApR) that do not harbor AsiSI sites.
Since the typical methylase selection did not yield any positive clones initially, a second cloning strategy was attempted by direct PCR with primers based on the conserved regions of C5 methylases. There are ten highly conserved amino acid regions within most of C5 methylases. Degenerate primers were synthesized based on the conserved motifs I and VI. PCR was attempted to amplify part of the asiSIM gene. Three PCR products of different sizes were found, gel-purified, and sequenced, but none of them contained conserved C5 methylase motifs.
A third successful cloning strategy involved construction of a vector with pUC19 origin and KmR, containing a single AsiSI site within the Km resistance gene (pUCKm). It was expected that modification of the AsiSI site within the KmR gene would render the plasmid intact following AsiSI digestion, thus giving rise to KmR colonies. If the AsiSI site within the KmR gene is not modified, the plasmid would be cleaved by AsiSI and lost during transformation. As a result, no KmR colony formation would be detected. The KmR selection marker also solved the problem of contamination by ApR cloning vectors. NlaIII partial genomic DNA library was constructed and challenged with AsiSI or SgfI (SgfI is an isoschizomer of AsiSI). Following methylase gene selection four AsiSI-resistant clones were identified. DNA sequencing indicated that the cloned methylase gene encoded a C5 methylase and displayed high homology to other C5 methylases.
Since restriction genes are usually located in close proximity with methylase gene, inverse PCR was employed to clone the adjacent DNA surrounding the asiSIM gene. Open reading frames (ORF) were identified on both sides of the asisIM gene. The downstream ORF was found to be homologous to a pilus assembly protein, thus it was predicted that this was not the asiSIR gene. The upstream ORF did not show significant homology to any gene in Genbank. In the first expression strategy, both the asiSIR and asiSIM genes were amplified by PCR and cloned in a T7 expression vector. Clones with inserts were identified, but no AsiSI activity was detected in cell extract. The second expression strategy used two-plasmid expression system. The asiSIM gene was first cloned in pACYC184 to premodify expression host ER2566, and the putative asiSIR gene was cloned in a T7 vector pET21at. AsiSI activity was detected in IPTG-induced cell extracts. This ORF was named as asiSIR. Three clones with high AsiSI activity were sequenced and one confirmed to be the wild type sequence.